The major aim of this research is to learn about the cellular, and ultimately the molecular, interactions that produce an ordered set of neuronal connections within the mammalian auditory periphery. Mammals possess two classes of auditory receptors, termed inner and outer hair cells, that project to the brain through separate neural components of the auditory nerve. Within the cochlea, auditory neurons innervate hair cells with astonishing accuracy; most form a single punctate synapse with only one receptor. Past work has shown that, during cochlear development, this neural specificity arises through extensive structural remodeling of individual cochlear neuron arbors, most of which form transient, supernumerary connections with outer hair cell receptors. During this process, up to 30% of all cochlear neurons die within the neonatal ear. This application, for renewal of a project in its 6th year, proposes to continue investigations into the formation of cochlear innervation by examining the cellular interactions between developing auditory neurons and hair cells, in-vivo, within the developing gerbil and, in vitro, within developing organotypic cultures of gerbil cochlea. Three sets of experiments are proposed: 1) The timecourse and spatial distribution of programmed auditory neuron death will be determined within the developing ear, by DNA labeling of apoptotic cells and serial reconstruction of the developing spiral ganglion; 2) Factors affecting the survival and programmed death of auditory neurons within the developing ear will be investigated by examining, both in vivo and in vitro, the cellular expression patterns of two cochlear neurotrophic molecules (BDNF and NT-3) and their receptors (trk B and trkC) using in situ hybridization and immunocytochemistry. 3) The influence of these neurotrophic factors on the formation of neural connections to inner and outer hair cells will be assessed by examining the structural development of individual auditory neuron arbors, labeled with neural tracers and reconstructed by light and confocal microscopy, within organotypic cultures that have been enriched for NT-3 and/or BDNF or depleted of these neurotrophins by the addition of trkB-IgG and trkC-IgG fusion proteins. Eighty-percent of all significant hearing impairment in the U.S is caused by the permanent loss of auditory neurons and receptors. It is crucial to understand the cellular interactions that form and maintain these sensorineural elements if effective biological strategies are to be devised for their protection or repair.